The present invention is directed, in part, to methods of using elongation factor p (efp) and related constituents of ribosomal complexes which comprise efp, the 50S ribosomal subunit, the 30S ribosomal subunit, the 70S ribosome, and related proteins, cofactors and enzymes to identify compounds that modulate prokaryotic cell function. Antibiotic compounds affecting such cell functions and methods of using those compounds to treat microbial infections in mammals are also described.
An important catalytic function of ribosomes is the synthesis of peptide bonds. Various studies have suggested that the 70S ribosome, which is comprised of the 30S and 50S ribosomal subunits, is important for protein synthesis.
Models of protein synthesis assume that once the aminoacyl-tRNA is bound to the ribosomal A-site of the 70S ribosome complex, peptidyltransferase, an integral part of the 50S ribosomal subunit, can condense all twenty-one aminoacyl-tRNAs with equal efficiency, without intervention of exogenous proteins and GTP. However, several studies indicate that in vitro, the peptidyltransferase condenses predominantly hydrophobic amino acids. Peptide bond synthesis in vitro is also dependent upon aminoacyl moieties. In particular, prokaryotic 70S ribosomes cannot efficiently incorporate certain amino acids into polypeptides from cytidyl aminoacyl-adenosine (CA) (analogues of the 3xe2x80x2-terminal end of aminoacyl-tRNAs). As well, several antibiotics, such as anisomycin and chloramphenicol, inhibit peptide bond synthesis with some aminoacyl-tRNAs and not with others.
A prokaryotic gene encoding one of several known types of elongation factor proteins, elongation factor p (efp) was cloned and sequenced. Aoki et al., Nucleic Acids Research, 1991 (19), pp. 6215-6220, the disclosure of which is incorporated herein by reference in its entirety. Efp has been found to be essential for cell viability. Efp stimulates the efficiency of the peptidyltransferase activity of procaryotic ribosomes between fMet-tRNAfMet and analogues of various aminoacyl-tRNAs. For example, the Kxe2x80x2 for the cytidyl(3xe2x80x2-5xe2x80x2)-[2xe2x80x2(3xe2x80x2)-O-L-CA-Gly is enhanced 50-fold, whereas that for CA-Phe is essentially unaltered by efp. Efp may modulate the efficiency of protein synthesis by controlling the rate of synthesis of certain peptide bonds. There are 800-900 molecules of efp per E. coli, or about 0.1 to 0.2 copy per ribosome, suggesting that efp may function catalytically in the cell. The preparation and isolation of efp can be found in M. C. Ganoza et al., Eur. J. Biochem., 1985, vol. 146, pp. 287-294, and/or D-G. Chung et al. Chapter 4, pp. 69-80 of Ribosomes and Protein Synthesis, A Practical Approach, edited by G. Spedding, 1990, IRL Press at Oxford University Press, Oxford, N.Y. and Tokyo, the disclosures of which are incorporated herein by reference in their entirety.
The requirements of peptide-bond and ester-bond formation stimulated by efp have been studied with fMet-tRNAfMet bound to 30S subunits and native or reconstituted 50S subunits. Efp functions in both peptide- and ester-bond synthesis promoted by the peptidyltransferase. The L16 protein (N-terminal fragment) of the 50S subunit is required for the efp-mediated synthesis of peptide bonds, whereas the L11, L15, and L7/L12 are not required in this reaction, suggesting that efp may function at a different ribosomal site than most other translation factors.
Of interest is the fact that efp differentially stimulates peptide bond synthesis when various amino acids are covalently linked to aminoacyl-adenosine (CA). It is possible that efp preferentially acts on weak acceptors for the peptidyltransferase. The specific mechanism whereby efp stimulates bond synthesis is not entirely clear. Efp may help accommodate fMet-tRNAfMet or peptidyl-tRNAs, or both, within the active center of the peptidyltransferase or it could affect peptidyltransferase directly.
The position occupied by each species of aminoacyl-tRNA on the ribosomes has been studied using antibiotics that are known to inhibit specific sites on the ribosome. Two types of A sites can be distinguished by their different reactivities towards specific antibiotics. The first site (of the i type) occur after fMet-tRNAfMet has directly entered the ribosomal P-site, where the E-site is free. The second A-site (of the e type) is the one normally used to bind aminoacyl-tRNAs to 70S ribosomes during the course of chain elongation.
The antibiotics neomycin, thiostrepton, and hygromycin appear to inhibit translocation and occupation of the A site, but they inhibit only about 20% or have no effect on efp reaction. These antibiotics also have no effect on formation of the fMet-tRNAfMet/ribosome translation complex nor on the peptide-bond synthesis which occurs in the absence of efp.
Streptomycin at 2xc3x9710xe2x88x925 M, which causes misreading and also inhibits A-site occupation of the e type, is a potent inhibitor of the efp-mediated reaction. The efp-mediated reaction is one in which purified efp is added to a translation complex of fMet-tRNAfMet: 70S ribosome:mRNA; and then puromycin or an appropriate amino acid-charged tRNA is added. Efp mediates the formation of a peptide bond between the fmet and the second amino acid. Streptomycin is known to interact with two sites on the 16S rRNA of the 30S subunit. It is unknown, however, whether streptomycin acts to directly inhibit efp.
Lincomycin inhibits peptidyltransferase and occupation of the A-site of the e type. Lincomycin has marginal effects on the synthesis of polyphenylalanine, but inhibits the puromycin reaction and nullifies the ability of efp to stimulate synthesis of peptide bonds. Erythromycin, also inhibits peptidyltransferase and it destabilizes the peptidyl-tRNA/ribosome/mRNA complex but it has no apparent effect on the efp reaction at 5xc3x9710xe2x88x925.
The present invention involves the surprising discovery of the critical role that efp may have in the procaryotic cell, and its role as a key component in the search for novel antimicrobial agents. These and other aspects of the invention are described below.
There is a need for more rapid and direct methods to screen compounds which may modulate ribosome mediated peptide bond formation. Such screening assays may discover new and useful antibiotics. New screens to detect and characterize compounds that affect efp and its functioning in the 70S ribosome, the 50S and 30S ribosomal subunits, and related proteins are disclosed herein. Newly discovered compounds or agents may promote cell death. The new understanding of the mechanism of action of known antimicrobials disclosed here may extend the usefulness of those antimicrobial agents.
Because of the surprising discovery disclosed here for the critical role that efp plays in the procaryotic cell, we can now disclose several aspects of this invention. Described herein are new methods or procedures to screen for, detect and/or characterize new compounds that modulate the function of efp in the prokaryotic cell. These methods or procedures include new in vitro methods as well as new in vivo methods.
In some embodiments of the invention, methods for identifying a compound which modulates activity of a prokaryotic elongation factor p in an in vitro assay, a cell based assay to determine the affect of the compound on cell function, a cell free extract assay to determine the affect of the compound on cell function are provided. The in vitro assay preferably comprises the steps of exposing elongation factor p with a compound, determining whether the compound modifies activity of the elongation factor p, and the cell-based assay preferably comprises determining whether the compound modifies activity of cell function. In some embodiments of the invention, the in vitro assay comprises determining whether the activity of the elongation factor p is decreased, determining whether the elongation factor p binds to the compound, determining whether the compound interferes with a function of the elongation factor p, or determining whether the compound interferes with a protein essential to the function of the elongation factor p such as the protein known as L16, or determining whether the compound binds to the ribosome or some component thereof that prevents the binding of efp to the ribosome and therefore interferes with the proper functioning of efp. In some embodiments of the invention, the step comprises measuring association of the compound with elongation factor p. In some embodiments of the invention, disclosures are to methods for determining whether a compound decreases a function of the cell.
Also disclosed herein are new methods or procedures to screen for, detect and/or characterize new compounds that modulate the function of the 30S ribosomal subunit when it interacts with efp in the prokaryotic cell. These methods or procedures include new in vitro methods as well as new in vivo methods.
In some embodiments of the invention, disclosures are to methods for determining whether a compound modulates the function of the 30S ribosome, this can be accomplished in a variety of ways, including but not limited to determining whether the compound inhibits binding of fmet-tRNA or mRNA to the 70S ribosome; determining whether the compound prevents the 50S subunit from binding to the 30S subunit, thereby preventing formation of a functional 70S ribosome; determining whether the compound inhibits the binding of any aminoacyl-tRNA to the ribosome; and determining whether a compound prevents the binding of initiation factor 1, initiation factor 2, initiation factor 3, or other factors necessary for formation of the initiation of the initiation complex or first peptide bond synthesis. In some embodiments of the invention, under in vitro conditions, the third step comprises measuring the presence of initiation complex in the cell, wherein a decrease in the amount of the complex confirms that the compound interferes with the interaction of efp and the 30S ribosome. In some embodiments, the third step comprises measuring affinity or displacement of fmet-tRNA to the complex, wherein a low affinity indicates that the compound interacts with the 30S complex containing elongation factor p. Following these procedures the compounds can then be exposed to cell based assays to determine the viability of the cells treated with the compounds.
Also disclosed herein are new methods or procedures to screen for, detect and/or characterize new compounds that modulate the function of the 50S ribosomal subunit when it interacts with efp in the prokaryotic cell. These methods or procedures include new in vitro methods as well as new in vivo methods.
In some embodiments of the invention, disclosures are to methods for determining whether a compound modulates the function of the 50S ribosome, this can be accomplished in a variety of ways, including but not limited to determining whether the compound inhibits binding of fmet-tRNA or mRNA to the 70S ribosome; determining whether the compound inhibits formation of the first peptide bond between fmet and the second amino acid; determining whether the compound prevents the 50S subunit from binding to the 30S subunit, thereby preventing formation of a functional 70S ribosome; determining whether the compound inhibits the binding of any aminoacyl-tRNA to the ribosome; and determining whether a compound prevents the binding of initiation factor 1, initiation factor 2, initiation factor 3, or other factors necessary for formation of the initiation of the initiation complex or first peptide bond synthesis. In some embodiments of the invention, under in vitro conditions, the third step comprises measuring the presence of initiation complex in the cell, wherein a decrease in the amount of the complex confirms that the compound interferes with the interaction of efp and the 50S ribosome. In some embodiments of the invention, the third step comprises measuring affinity or displacement of fmet-tRNA to the complex, wherein a low affinity indicates that the compound interacts with the 50S complex containing elongation factor p. Following these procedures the compounds can then be exposed to cell-based assays to determine the viability of the cells treated with the compounds.
Also disclosed herein are new methods or procedures to screen for, detect and/or characterize new compounds that modulate the function of the 70S ribosome when it interacts with efp in the prokaryotic cell. These methods or procedures include new in vitro methods as well as new in vivo methods.
In some embodiments of the invention, disclosures are to methods for determining whether a compound modulates the function of the 70S ribosome, this can be accomplished in a variety of ways, including but not limited to including but not limited to determining whether the compound inhibits binding of fmet-tRNA or mRNA to the 70S ribosome; determining whether the compound inhibits formation of the first peptide bond between fmet and the second amino acid; determining whether the compound prevents the 50S subunit from binding to the 30S subunit, thereby preventing formation of a functional 70S ribosome; determining whether the compound inhibits the binding of any aminoacyl-tRNA to the ribosome; and determining whether a compound prevents the binding of initiation factor 1, initiation factor 2, initiation factor 3, or other factors necessary for formation of the initiation of the initiation complex or first peptide bond synthesis. In some embodiments of the invention, the third step comprises measuring the presence of initiation complex, wherein a decrease in the amount of the complex confirms that the compound interferes with the interaction of efp and the 70S ribosome. In some embodiments of the invention, the third step comprises measuring affinity or displacement of fmet-tRNA to the complex, wherein a low affinity indicates that the compound interacts with the 70S complex containing elongation factor p. Following these procedures the compounds can then be exposed to cell-based assays to determine the viability of the cells treated with the compounds.
In some embodiments of the invention a eukaryote protein known as eIF5A, a protein that performs a similar function in eukaryote cells as the efp protein does in prokaryotic cells is used to further identify and characterize compounds that have little or no modulating effect or inhibiting effect on eIF5A but that do modulate or inhibit efp. New methods or procedures, that employ all of the methods described above, to detect or characterize a new compound are then combined with the additional steps of determining that compounds modulating activity on the eukaryote version of efp (or eIF5A) and then comparing the modulating activity of the newly identified compound on prokaryotic efp with its modulating activity on eIF5A in order to identify or characterize compounds that inhibit efp activity on prokaryotic cells but have little adverse affect on the activity of eIF5A.
Also disclosed herein are new methods or procedures that allow the inhibition or control the growth of microbial organisms. Methods which disclose compounds that modulate the function of efp in the prokaryotic cell.
One important aspect of this invention is the discovery of the use of known compounds that can now be used to interfere with the important function of the efp protein.
The present invention further provides methods of modulating the activity of a bacterial elongation factor p comprising contacting the protein or a cell containing the protein with an oxazolidinone compound.
The present invention further provides methods of modulating the activity of a bacterial 30S ribosomal subunit comprising contacting the protein or a cell containing the subunit with an oxazolidinone.
The present invention further provides methods of modulating the activity of a bacterial 50S ribosomal subunit comprising contacting the protein or a cell containing the subunit with an oxazolidinone.
The present invention further provides methods of modulating the activity of a bacterial 70S ribosome comprising contacting the protein or a cell containing the subunit with an oxazolidinone.
The present invention further provides methods of modulating the activity of a bacterial L16 protein comprising contacting the protein or a cell containing the subunit with an oxazolidinone.
The present invention further provides methods of modulating the activity of a bacterial elongation factor p comprising contacting the protein or a cell containing the protein with an oxazolidinone type of compound.
These and other aspects of the invention are described in greater detail below.
Various definitions are made throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of invention as a whole and as are typically understood by those skilled in the art.
As used herein, the term xe2x80x9cactivityxe2x80x9d refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e. having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of the compound for directly binding efp or aribosome, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event.
As used herein, the term xe2x80x9caffectsxe2x80x9d means either a decrease or increase in the amount or quality of a particular cell function in response to some stimulus, exposure or event.
As used herein, the term xe2x80x9cbindingxe2x80x9d means the physical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or because of another protein or compound. Direct binding refers to interactions that do not take place through or because of another protein or compound but instead are without other substantial chemical intermediates.
As used herein, the phrase xe2x80x9ccell functionxe2x80x9d is defined to include all aspects of functionality of cells including cell viability but, especially, cell growth.
As used herein, the term xe2x80x9ccompoundxe2x80x9d means any identifiable chemical or molecule, small molecule, peptide, protein, sugar, natural or synthetic, or a discrete agent such as a specific amount of light, energy or temperature that is suspected to potentially interact with the process or system of interest, here typically efp, 30S, 50S, 70S ribosomes and related proteins.
As used herein, the term xe2x80x9ccontactingxe2x80x9d means either direct or indirect, in vitro or in vivo administration of a compound to target, where the target may be a protein, ribosome, portion of a cellular system, whole cell, tissue, or mammal. The target may be in an in vitro or in vivo system with any number of buffers, salts, solutions etc.
As used herein, the phrases and terms xe2x80x9celongation factor pxe2x80x9d, xe2x80x9cefpxe2x80x9d, xe2x80x9cef-pxe2x80x9d, xe2x80x9cEFPxe2x80x9d, or xe2x80x9cEF-Pxe2x80x9d refers to the prokaryotic protein having a function like that of the efp protein isolated from E. coli or various other bacteria or recombinant versions of that protein such as that described by M. C. Ganoza et al., Eur. J Biochem 1985, vol. 146, pp. 287-294, and H. Aoki et al. Biochimie (1997) vol.79, pp. 7-11, Aoki et al., Nucleic Acids Research, 1991 (19), pp.6215-6220, the disclosures of which are incorporated herein by reference in their entirety. The nucleic acid sequence of one or more of such proteins are provided in the references and the specification below. The EF-P differs from EF-Tu and EF-G in that it does not require GTP for its activity. Efp can be cloned, synthesized, or otherwise manipulated and if a version is made active according to any of the screens described here or in the references cited then that protein should be considered an efp protein.
As used herein, the term xe2x80x9ceffectsxe2x80x9d means either a decrease or increase in the amount or quality of a particular cell function in response to some stimulus, exposure or event.
As used herein, the phrase xe2x80x9cfirst peptide bond reactionxe2x80x9d means the joining of the I carboxyl group of formylmethionine to the I amino group of another amino acid.
As used herein, the phrase xe2x80x9cformation of the initiation complexxe2x80x9d means formation of a complex containing N formylmethionyl tRNA, 30S subunit, mRNA, GTP and the initiation factors IF1, IF2 and IF3.
As used herein, the term xe2x80x9cinteractingxe2x80x9d means direct binding, including selective or specific binding, to a constituent of the ribosomal complex such that cell function is effected.
As used herein, the term xe2x80x9cL16xe2x80x9d means the L16 prokaryotic protein involved in bacterial protein synthesis as described in H. Aoki, et al., Molecular Characterization of the Procaryotic Efp Gene Product Involved in a Peptidyltransferase Reaction, Biochimie (1997) vol. 79, pp. 7-11, the disclosure of which is incorporated herein by reference in its entirety.
As used herein, the terms xe2x80x9cmodulatesxe2x80x9d or xe2x80x9cmodifiesxe2x80x9d means an increase or decrease in the amount, quality, or effect of a particular activity or protein.
As used herein, the phrase xe2x80x9c70S ribosomexe2x80x9d means a prokaryotic ribonucleoprotein particle with a sedimentation coefficient of 70S that can be dissociated into a large subunit of 50S and a small subunit of 30S.
As used herein, the phrase xe2x80x9c50S ribosomexe2x80x9d or xe2x80x9c50S subunitxe2x80x9d means a prokaryotic ribonucleoprotein particle with a sedimentation coefficient of 50S that can be dissociated from a 70S ribosome.
As used herein, the phrase xe2x80x9c30S ribosomexe2x80x9d or xe2x80x9c30S subunitxe2x80x9d means a prokaryotic ribonucleoprotein particle with a sedimentation coefficient of 30S that can be dissociated from a 70S ribosome.
As used herein, the phrase xe2x80x9cpeptide bond donorxe2x80x9d means any compound that has a free amino group capable of forming a peptide bond with an amino acid. Preferred peptide bond donors include, but are not limited to, puromycin or a puromycin analog, or any amino acyl-tRNA or an analog of amino acyl-tRNA.
As used herein, the term xe2x80x9coxazolidinonexe2x80x9d means a compound of the class known as oxazolidinones, including the compounds described in U.S. Ser. Nos. 07/438,759, 07/553,795, 08/006,596, 07/882,407, 07/786,107, 07/831,213, 08/233,903, 08/119,279, 08/226,158, 08/155,988, 08/329,717, 07/909,387, 08/339,979, 08/384,278, 08/875,800, 07/880,432, 08/610,031, 08/332,822, 07/988,589, 08/003,778, 08/066,356, 08/438,705, 60/015,499, 60/003,149, 09/138,205, 09/138,209, 08/696,313, 60/012,316, 08/803,469, 60/003,838, 08/709,998, 60/008,554, 08/762,478, 60/007,371, 08/850,424, 60/048,342, 09/080,751, 60/052,907, 60/064,746, 09/111,995, 60/064,738, 60/065,376, 60/067,830, 60/089,498, 60/100,185, 09/081,164, 60/088,283, 60/092,765, 07/244,988, 07/253,850; European Patents EP 0500686, EP 0610265, EP 0673370; PCT Application Numbers PCT/US90/06220, PCT/US94/08904, PCT/US94/10582, PCT/US95/02972, PCT/US95/10992, PCT/US93/04850, PCT/US95/12751, PCT/US96/00718, PCT/US93/03570, PCT/US93/09589, PCT/US96/05202, PCT/US97/03458, PCT/US96/12766, PCT/US97/01970, PCT/US96/14135, PCT/US96/19149, PCT/US96/17120, PCT/US98/09889, PCT/US98/13437; and U.S. Pat. Nos. 5,700,799, 5,719,154, 5,547,950, 5,523,403, 5,668,286, 5,652,238, 5,688,792, 5,247,090, 5,231,188, 5,654,428, 5,654,435, 5,756,732, 5,164,510, 5,182,403, 5,225,565, 5,618,949, 5,627,197, 5,534,636, 5,532,261, 5,776,937, 5,529,998, 5,684,023, 5,627,181, 5,698,574, 5,220,011, 5,208,329, 5,036,092, 4,965,268, 4,921,869, 4,948,801, 5,043,443, 5,130,316, 5,254,577, 4,877,892, 4,791,207, 4,642,351, 4,665,171, 4,734,495, 4,775,752, 4,870,169, 4,668,517, 4,340,606, 4,362,866, 4,193,918, 4,000,293, 3,947,465, 4,007,168, 3,674,780, 3,686,170, 3,906,101, 3,678,040, 3,177,114, 3,141,889, 3,149,119, 3,117,122, 5,719,154, 5,254,577, 4,801,600, 4,705,799, 4,461,773, 4,243,801, 3,794,665, 3,632,577, 3,598,830, 3,513,238, 3,598,812, 3,546,241, 3,318,878, 3,322,712; the disclosures of which are incorporated herein by reference in their entirety. Preferred oxazolidinones include linezolid and eperezolid.
The description of this invention is organized into several parts. The different parts and not exclusive of each other, they all describe one invention but different aspects and applications of the invention will be emphasized and described in greater or lesser detail in the different parts of the description. One part will emphasize the methods and procedures whereby efp is used as molecular target to find, identify or characterize compounds that modulate the activity of efp, especially compounds that interfere or inhibit that activity. A subportion describes in vitro methods of evaluating efp and in vivo applications thereof. Other parts are structured similarly to the first part and conceptually should include the first part, only emphasis and methods directed to the 30S ribosomal subunit, the 50S ribosomal subunit, and the 70S ribosome are described in these other parts. Another part refers to and includes using the methods and procedures and the information in the other parts and applying it in a novel fashion, which is to add the additional procedure of comparing the information of the first parts to with similar information about the compounds identified from steps previously identified to a similar study of the activity of those compounds on eIF5A. Another part describes compounds that are now known to have expected activity against the prokaryotic functions and systems described in the previously described parts.
The present invention is directed, in part, to methods for identifying compounds which modulate activity of efp or translation initiation complex when interacting with efp. In addition, the methods of the present invention also include, in a similar manner, identifying compounds which modulate activity of prokaryotic 30S subunit, 50S subunit, and 70S subunit of the ribosome.
Efp, as well as the other components described above, can be isolated from a natural source such as, for example, a bacteria, like E. coli or various other bacteria, such as, for example S. aureus, S. pneumoniae, H. influenzae, and an Enterococcus species. In addition, recombinant versions of these proteins, such as that described by M. C. Ganoza et al, Eur. J Biochem 1985, vol. 146, pp.287-294, and H. Aoki et al. Biochimie (1997) vol. 79, pp. 7-11, Aoki et al., Nucleic Acids Research, 1991 (19), pp. 6215-6220, the disclosures of which are incorporated herein by reference in their entirety, can be prepared. One skilled in the art is readily able to prepare such recombinant proteins.
In a preferred embodiment the efp can be a recombinant protein having post-translation modifications, such as, for example, those modifications selected from the group consisting of efp proteins where the lysine residues are modified. Recombinant proteins can be prepared in eukaryotic systems such as, for example, using the baculovirus expression vectors which are well known to the skilled artisan.
The preferred form of efp is the native form of the protein purified from S. aureus, E. coli or other pathogenic bacteria. However, according to the present invention, other forms of efp include the native form of the protein purified from various gram positive bacterial pathogens, including: Staphylococcus aureus; Staphylococcus epidermidis (A, B, C biotypes); Staphylococcus caseolyticus; Staphylococcus gallinarum; Staphylococcus haemolyticus; Staphylococcus hominis; Staphylococcus saprophyticus; Streptococcus agalactiae (group B); Streptococcus mutans/rattus; Streptococcus pneumoniae; Streptococcus pyogenes (group A); Streptococcus salivarius; Streptococcus sanguis; Streptococcus sobrinus; Actinomyces spps.; Arthrobacterhistidinolovorans; Corynebacterium diptheriae; Clostridium difficle; Clostridium spps.; Enterococcus casseliflavus; Enterococcus durans; Enterococcus faecalis; Enterococcus faecium; Enterococcus gallinarum; Erysipelothrix rhusiopathiae; Fusobacterium spps.; Listeria monocytogenes; Prevotella spps.; Propionibacterium acnes; and Porphyromonas gingivalis. 
Still other forms of efp include the native form of the protein purified from various gram negative bacterial pathogens, including: Acinetobacter calcoaceticus; Acinetobacter haemolyticus; Aeromonas hydrophila; Bordetella pertussis; Bordetella parapertussis; Bordetella bronchiseptica; Bacteroides fragilis; Bartonella bacilliformis; Brucella abortus; Brucella melitensis; Campylobacter fetus; Campylobacter jejuni; Chlamydia pneumoniae; Chlamydia psittaci; Chlamydia trachomatis; Citrobacterfreundii; Coxiella burnetti; Edwardsiella tarda; Edwardsiella hoshinae; Enterobacter aerogenes, Enterobacter cloacae (groups A and B); Escherichia coli (to include all pathogenic subtypes) Ehrlicia spps.; Francisella tularensis; Haemophilus actinomycetemcomitans; Haemophilus ducreyi; Haemophilus haemolyticus; Haemophilus influenzae; Haemophilus parahaemolyticus; Haemophilusparainfluenzae; Hafnia alvei; Helicobacterpylori; Kingella kingae; Klebsiella oxytoca; Klebsiella pneumoniae; Legionella pneumophila; Legionella spps.; Morganella spps.; Moraxella cattarhalis; Neisseria gonorrhoeae; Neisseria meningitidis; Plesiomonas shigelloides; Proteus mirabilis; Proteus penneri; Providencia spps.; Pseudomonas aeruginosa; Pseudomonas species; Rickettsia prowazekii; Rickettsia rickettsii; Rickettsia tsutsugamushi; Rochalimaea spps.; Salmonella subgroup 1 serotypes (to include S. paratyphi and S. typhi); Salmonella subgroups 2, 3a, 3b, 4, and 5; Serratia marcesans; Serratia spps.; Shigella boydii; Shigellaflexneri; Shigella dysenteriae; Shigella sonnei; Yersinia enterocolitica; Yersiniapestis; Yersiniapseudotuberculosis; Vibrio cholerae; Vibrio vulnificus; and Vibrio parahaemolyticus. 
Still other forms of efp include the native form of the protein purified from various Mycobacterial species, including: Mycobacterium tuberculosis; Mycobacterium avium; and other Mycobacterium spps.
Still other forms of efp include the native form of the protein purified from various Mycoplasmas (or pleuropneumonia-like organisms), including: Mycoplasma genitalium; Mycoplasma pneumoniae; and other Mycoplasma spps.
Still other forms of efp include the native form of the protein purified from various Treponemataceae (spiral organisms), including: Borrelia burgdoreri; other Borrelia species; Leptospira spps.; Treponema pallidum. 
S. aureus efp is defined as a protein having an amino acid sequence with at least about 70% homology determined by, for example, alignment and direct one-to-one correspondence with the following protein sequence MISVNDFKTG LTISVDNAIW KVIDFQHVKP GKGSAFVRSK LRNLRTGAIQ EKTFRAGEKV EPAMIENRRM QYLYADGDNH VFMDNESFEQ TELSSDYLKE ELNYLKEGME VQIQTYEGET IGVELPKTVE LTVTETEPGI KGDTATGATK SATVETGYTL NVPLFVNEGD VLIINTGDGS YISRG (SEQ ID NO: 1) and having activity in the efp activity assay described below.
E. coli efp is defined as a protein having an amino acid sequence with at least about 70% homology, determined as described above, with the following sequence MATYYSNDFRA GLKIMLDGEP YAVEASEFVK PGKGQAFARV KLRRLLTGTR VEKTFKSTDS AEGADVVDMN LTYLYNDGEF WHFMNNETFE QLSADAKAIG DNAKWLLDQA ECIVTLWNGQ PISVTPPNFV ELEIVDTDPG LKGDTAGTGG KPATLSTGAV VKVPLFVQIG EVIKVDTRSG EYVSRVK (SEQ ID NO:2) and having activity in the efp activity assay described below.
In preferred embodiments, in order to identify compounds which modulate efp activity or activity of any of the other components of the ribosome described above, in vitro assays are disclosed, wherein, for example, cell-free extract comprising efp (as well as any of the other components described above; 30S, 50S, and 70S) and the translation initiation complex, or components thereof, is contacted with a test compound. The contacting can take place in buffers or media well known to those skilled in the art. In addition, varying amounts of the test compound can be used as desired by the practitioner. Test compounds provided herein, including those identified by the present methods, can be formulated into pharmaceutical compositions by, for example, admixture with pharmaceutically acceptable nontoxic excipients and carriers. Test compounds which test positive can be used as antiseptic agents. Accordingly, the methods of the present invention also include a method of identifying antiseptic agents.
In some embodiments of the invention, a method for identifying a compound which modulates the activity of prokaryotic efp comprises preparing a solution of efp; contacting the solution containing efp with the compound; and determining whether the compound modifies activity of efp. Whether the compound modifies the activity of the efp is determined by, for example, determining whether the compound binds to efp. Binding can be determined by employing a number of art-recognized procedures.
Determining whether the compound binds to efp can be accomplished by a binding assay including, but not limited to, gel-shift mobility electrophoresis, Western blot, filter binding, and scintillation proximity assay. U.S. Pat. No. 4,568,649, which is disclosed herein by reference in its entirety, teaches a scintillation proximity assay. Additional information regarding scintillation proximity assay systems and applications is available from Amersham Pharmacia Biotech (UK, Little Chalfont, Buckinghamshire, England HP79NA).
Determining whether the compound binds to efp can also be accomplished by measuring the intrinsic fluorescence of efp and determining whether the intrinsic fluorescence is modulated in the presence of the compound. Preferably, the intrinsic fluorescence of efp is measured as a function of the tryptophan residue(s) of efp. Preferably, fluorescence of efp is measured and compared to the fluorescence intensity of efp in the presence of the compound, wherein a decrease in fluorescence intensity indicates binding of the compound to efp. Preferred methodology is set forth in xe2x80x9cPrinciples of Fluorescence Spectroscopyxe2x80x9d by Joseph R. Lakowicz, New York, Plenum Press, 1983 (ISBN 0306412853) and xe2x80x9cSpectrophotometry And Spectrofluorometryxe2x80x9d by C. L. Bashford and D. A. Harris Oxford, Washington D.C., IRL Press, 1987 (ISBN 0947946691), the disclosures of which are incorporated herein by reference in their entirety.
In other embodiments of the invention, the method described above further comprises determining whether the compound interfering with the function of efp is interfering with other protein(s) essential for the functioning of efp. Preferably, the other protein essential for the functioning of efp is L16 protein.
In other embodiments of the invention, a method for identifying a compound which modulates the activity of prokaryotic efp comprises preparing a solution of efp; contacting the solution of efp with a radiolabeled oxazolidinone, isolating or measuring the radiolabeled oxazolidinone bound to efp; contacting the compound with the radiolabeled oxazolidinone bound to efp; and determining whether the compound displaces the radiolabeled oxazolidinone from efp. Additionally, the method may further comprise measuring the displacement of the radiolabeled oxazolidinone from efp. Preferably, determination of displacement is accomplished by comparing the amount of the detectable radiolabel in the solution prior to addition of the compound with the amount of detectable radiolabel in the solution after addition of the compound, wherein a decrease in detectable radiolabel indicates that the compound displaces the radiolabeled oxazolidinone compound from the complex. Radiolabeled competitive binding studies are described in A. H. Lin et al. Antimicrobial Agents and Chemotherapy, 1997, vol.41, no.10. pp.2127-2131, the disclosure of which is incorporated herein by reference in its entirety. Preferably, the radiolabeled oxazolidinone compound is linezolid or eperezolid.
In some embodiments, the activity of the efp-mediated activity (or activity of any of the other ribosomal components described above) can be measured by the amount of translation initiation complex formed. One skilled in the art is readily familiar with measuring the amount of translation initiation complex formed. A compound that inhibits efp will be reflected in the amount of translation initiation complex formed in vitro. Preferably, the method comprises preparing a first solution of efp; preparing a second solution comprising N-formylmethionyl-tRNA (fMet-tRNA), 30S subunit, 50S subunit, any mRNA containing an AUG sequence, and initiation factors 1, 2, and 3; contacting the second solution with the first solution and the compound; and determining whether the compound allows fMet-tRNA to bind to a complex formed through the interaction of efp, 30S subunit, 50S subunit, any mRNA containing an AUG sequence, and initiation factors 1, 2, and 3. Efp-mediated activity (or activity of any of the other ribosomal components described above) can be measured by measuring affinity or displacement of fMet-tRNA to said complex. A compound that inhibits the binding of fMet-tRNA to ribosomal complexes containing efp will be reflected in the amount of fMet-tRNA bound to the complex. The lower the amount of fMet-tRNA bound, the greater the inhibitory affect the test compound has. This type of affinity displacement is described by S. M. Swaney, et al. Antimicrobial Agents and Chemotherapy (1998) vol. 42, no. 12, pp.3251-3255, the disclosure of which is incorporated herein by reference in its entirety. Using ordinary skills and techniques in the art the procedures in this reference can be easily adapted to the invention described herein. Preferably, the mRNA containing an AUG sequence consists essentially of rArUrG. Preferably, efp is isolated from a natural source, such as a prokaryotic organism, preferably a bacteria including, but not limited to, E. coli, S. aureus, S. pneumoniae, H. influenzae, or an Enterococcus species. In all of the tests described herein, one skilled in the art can use fragments of mRNA of any length as long as the fragment comprises an AUG sequence.
In other embodiments of the invention, a method for identifying a compound which modulates the activity of efp comprises contacting a cell containing efp in vitro with a compound identified by the methods described above, and determining whether the compound inhibits cell growth. Alternately, the contacting can take place in vivo, in which an animal, such as, for example, a mammal or mouse or other suitable animal known to those skilled in the art, is contacted by administering a pharmaceutical composition comprising the test compound and pharmaceutically acceptable salt, carrier, or diluent. In addition, varying numbers of cells and concentrations of test compounds can be used. Whether the test compound increases or decreases activity of the efp is determined. In addition, whether the test compound promotes cell survival or cell death is also determined. The test compound can be administered to a mammal topically, intradermally, intravenously, intramuscularly, intraperitonally, subcutaneously, and intraosseously, or any other desired route and can be in any amount desired by the practitioner. Determination of the susceptibility of bacteria to particular compounds can be determined according to the methods described in National Committee for Clinical Laboratory Standards, 1993, Approved standard, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 3rd Ed., National Committee for Clinical Laboratory Standards, Villanova, Pa., the disclosure of which is incorporated herein by reference in its entirety.
In other embodiments of the invention, each of the above-described methods can be applied to compositions of efp which also contain either the 30S subunit, 50S subunit, or 70S ribosome. For example, in some embodiments of the invention, a method for identifying a compound which modulates the activity of prokaryotic efp comprises preparing a composition or solution of efp; adding prokaryotic 30S subunit (or 50S subunit or 70S ribosome) to the solution of efp; contacting the compound with the composition or solution of efp and 30S subunit (or 50S subunit or 70S ribosome); and determining whether the compound binds to efp in association with the 30S subunit (or 50S subunit or 70S ribosome) or whether the compound interferes with the binding of said efp and said 30S subunit (or 50S subunit or 70S ribosome). In some embodiments, determining whether the compound binds to efp in association with the 30S subunit (or 50S subunit or 70S ribosome) or whether the compound interferes with the binding of said efp and said 30S subunit (or 50S subunit or 70S ribosome) comprising determining whether the compound binds to the 30S subunit (or 50S subunit or 70S ribosome) or efp. In some embodiments, the intrinsic fluorescence of efp bound to said 30S subunit (or 50S subunit or 70S ribosome) is measured, as described above, and whether the intrinsic fluorescence is modulated by the compound is determined. Preferably, the intrinsic fluorescence of efp is measured as a function of changes in the fluorescence of the tryptophan residue(s) of efp, as described above. In addition, the above-described method may further comprise determining whether the compound interfering with the function of efp is interfering with other protein(s), such as L16 protein, essential for the functioning of efp. Determination of binding can be accomplished in the same manner as described above. In addition, competitive binding assays using a solution of efp and 30S subunit (or 50S subunit or 70S ribosome) with a radiolabeled oxazolidinone and a test compound can be performed essentially as described above.
In other embodiments of the invention, a method for identifying a compound which modulates the activity of prokaryotic efp comprises preparing a solution of radiolabeled efp; adding a 30S subunit (or 50S subunit or 70S ribosome) and the compound with the solution of radiolabeled efp; measure whether the 30S subunit (or 50S subunit or 70S ribosome) is bound to radiolabeled efp; and if the 30S subunit (or 50S subunit or 70S ribosome) is not bound to efp, then select the compounds which interfered with the binding thereof. Preferably, determination of binding is accomplished by employing a binding assay described above.
In other embodiments of the invention, a method for identifying a compound which modulates the activity of prokaryotic efp comprises preparing a first solution of efp;
preparing a second solution comprising N-formylmethionyl-tRNA (fMet-tRNA), 30S subunit (or 50S subunit or 70S ribosome), any mRNA containing an AUG sequence, and initiation factors 1, 2, and 3; and contacting the second solution with the first solution and the compound; and determining whether the compounds allows fMet-tRNA to bind to a complex formed through the interaction of efp, 30S subunit (or 50S subunit or 70S ribosome), any mRNA containing the AUG sequence, and initiation factors 1, 2, and 3. Preferably, the mRNA containing an AUG sequence consists essentially of rArUrG. Preferably, efp is isolated from a natural source, such as a prokaryotic organism, such as a bacteria including, but not limited to, E. coli, S. aureus, S. pneumoniae, H. influenzae, and an Enterococcus species.
In other embodiments of the invention, a method for identifying a compound which modulates the activity of prokaryotic efp comprises contacting a cell containing said efp and also the 30S subunit, 50S subunit or 70S ribosome with a compound identified by the previously described methods, and determining whether the compound inhibits cell growth, as described above.
In another embodiment, a method for identifying a compound which modulates the activity of prokaryotic efp comprises preparing a first solution of efp; preparing a second solution comprising 50S subunit or 70S ribosome, a tRNA fragment comprising CACCA-radiolabeled amino acid, and a peptide bond donor; contacting the second solution with the first solution and the compound; and determining whether the compound inhibits the first peptide bond reaction of a complex formed through the interaction of efp, 50S subunit or 70S ribosome, a tRNA fragment comprising CACCA-radiolabeled amino acid, and a peptide bond donor efp. Alternatively, the second solution can comprise N-formylmethionyl-tRNA (fMet-tRNA), 30S subunit, 50S subunit, any mRNA containing an AUG sequence, and initiation factors 1, 2, and 3, and a peptide bond donor, and it is determined whether the compound inhibits the first peptide bond reaction of a complex formed through the interaction of efp, fMet-tRNA, 30S subunit, 50S subunit, any mRNA containing an AUG sequence, and initiation factors 1, 2, and 3. A compound that inhibits efp (or any of the other ribosomal components described above) will be reflected in the amount of first peptide bond synthesis formed in vitro. For example, efp allows formation of a peptide bond between N-formylmethionine and the second amino acid (or puromycin as a substitute). Addition of a compound that inhibits the action of efp will prevent formation of the peptide bond, leaving the preformed initiation complex fMet-tRNA:70S ribosome:mRNA intact. M. C. Ganoza et al., Eur. J Biochem 1985, vol. 146, pp. 287-294, and/or D-G. Chung et al. Chapter 4, pp. 69-80 of Ribosomes and Protein Synthesis, A Practical Approach, edited by G. Spedding, 1990, IRL Press at Oxford University Press, Oxford, N.Y. and Tokyo, the disclosures of which are incorporated herein by reference in their entirety. The first peptide bond formation can also be determined according to the methods described in Monro, et al., J. Mol. Biol., 1967, 25, 347-350, Monro, et al., Methods Enzymol., 1971, 20, 472-481, the disclosures of which are incorporated herein by reference in their entirety. Preferably, the peptide bond donor includes, but is not limited to, puromycin and analogs thereof, and any amino acyl-tRNA and analogs thereof. Preferably, efp is isolated from a natural source, such as, for example, a prokaryotic organism, preferably, a bacteria, such as, for example, E. coli, S. aureus, S. pneumoniae, H. influenzae, or an Enterococcus species. The above described method can also be employed to identify a compound which inhibits the first peptide bond reaction of a complex formed through the interaction of efp, 50subunit or 70S ribosome, a tRNA fragment comprising CACCA-radiolabeled amino acid, and a peptide bond donor and efp. The above described method can also be employed to identify a compound which inhibits the first peptide bond reaction of a complex formed through the interaction of fMet-tRNA, 30S subunit, 50S subunit, any mRNA containing an AUG sequence, and initiation factors 1, 2, and 3, and a peptide bond donor and efp. In addition, one skilled in the art can determine whether the compound, identified as described above, inhibits cell growth by contacting a cell containing efp, or any other subunits or proteins described above.
In another embodiment, a method for identifying a compound which modulates the activity of prokaryotic efp comprises contacting a cell or solution containing efp with a detectably labeled oxazolidinone compound known to bind efp under conditions whereby efp forms a complex with the oxazolidinone compound; contacting the solution or cell with an unlabeled compound; and determining whether the unlabeled compound displaces the labeled oxazolidinone compound from the complex. Preferably, the cell or solution contains an oxazolidinone compound, a compound with a substantial binding affinity for efp, 30S, 50S, or 70S, L16 protein, or other components of the ribosome. Once the cell, or cell extract, or solution containing the components, is contacted with the test compound, the ribosomal complex containing the efp (and/or the other ribosome components described above) is isolated, and it is determined to what extent the test compound has displaced oxazolidinone. There are many techniques known in the art for determining displacement. Preferably, determination of displacement is accomplished by comparing the amount of the detectable label in the cell or solution prior to addition of the unlabeled compound with the amount of detectable label in the cell or solution after addition of unlabeled compound, wherein a decrease in detectable label indicates that the compound displaces the oxazolidinone compound from the complex. Preferably, the detectable label is a radiolabel or a fluorescent label. Radiolabeled competitive binding studies are described in A. H. Lin et al. Antimicrobial Agents and Chemotherapy, 1997, vol. 41, no. 10. pp. 2127-2131, the disclosure of which is incorporated herein by reference in its entirety. Fluorescent labeling is well know to the skilled artisan. Preferably, the oxazolidinone compound is linezolid or eperezolid.
In another embodiment of the invention, the test compound is further examined to determine whether it modulates the eukaryotic homolog of elongation factor p, eIF5A. eIF5A is described in Smit-McBride et al., Sequence Determination and cDNA Cloning of Eukaryotic Initiation Factor 4D, the Hypusine-containing Protein, (1989) J. Biol. Chem., vol. 264, pp. 1578-1583, the disclosure of which is incorporated here in by reference in its entirety. A method for identifying a compound which modulates the activity of prokaryotic efp but not eukaryotic eIF5A preferably comprises initially determining whether the compound modulates the activity of prokaryotic efp by any of the methods described herein; followed by the steps of preparing a first composition or solution of eIF5A; preparing a second solution comprising methionyl-tRNA (Met-tRNA), 80S ribosome, any mRNA containing an AUG sequence, initiation factors eIF-2, eIF-3, eIF-5, eIF-4C, eIF-4D, and a peptide bond donor; contacting the second solution with the first solution and the compound; and determining whether the compound inhibits the first peptide bond reaction of a complex formed through the interaction of eIF5A, Met-tRNA, 80S ribosome, any mRNA containing an AUG sequence, and initiation factors eIF-2, eIF-3, eIF-5, eIF-4C, eIF-4D, as described above. Preferred peptide bond donors include, but are not limited to, puromycin and analogs thereof, and any amino acyl-tRNA and analogs thereof. Preferred mRNA sequences must include rArUrG, but may include additional nucleotides. Preferably, eIF5A is isolated from a natural source, such as a eukaryotic organism, preferably a mammal.
In some embodiments of the invention, this determination is made in a manner similar to the determination for prokaryotic efp except eukaryotic eIF5A is used. Preferably, compounds which modulate prokaryotic efp but not eukaryotic eIF5A are identified as described in K. Moldave, Eukaryotic Protein Synthesis, (1985) Ann. Rev. Biochem, vol.54, pp. 1109-1149, the disclosure of which is incorporated herein by reference in its entirety. In addition, first peptide bond formation can be analyzed as described in Benne et al., J. Biol. Chem., 1978, 253, 3070-3087, the disclosure of which is incorporated herein by reference in its entirety.
The present invention is further directed to methods of modulating the activity of efp, 30S subunit, 50S subunit, or 70S subunit, L16 protein, or ribosomal subunits containing any of the same, or cells or cell preparations (including cell lysates) containing any of the same by contacting any of the above-described samples with an oxazolidinone compound. Contacting can be in vitro or in vivo by any of the routes of administration described above. The oxazolidinone can be formulated as described above into a pharmaceutical composition.